This invention is particularly concerned with the identification of unknown substances and the recognition of trace compounds which are separated by different means:
a) by HPLC (High Performance Liquid Chromatography) and detected post column on small bore activated or inactivated capillaries at the outlet of the column, PA1 b) by micro-HPLC (High Performance Liquid Chromatography) and detected on column in a narrow bore capillary, PA1 c) by CE (Capillary Electrophoresis) or CEC (Capillary Electro Chromatography) and detected on column. PA1 maximizing the irradiated volume, PA1 maximizing the collection efficiency, PA1 maintaining the best separation efficiency (variance due to the detection), and PA1 maintaining the lowest laser light scattering. PA1 c is the fluorophor concentration PA1 .epsilon. is the molar absorption factor of the fluorophor PA1 .phi. is the fluorescence quantum efficiency of the fluorophor. PA1 P is the power of the light exciting the molecules in the internal diameter of the capillary. PA1 S is the section of the light beam exciting the molecules PA1 reflections on the optical elements, including the silica capillary walls, PA1 scattering due to the buffers flowing in the capillary, PA1 physical phenomena such as Raman scattering. PA1 means for emitting a laser beam, PA1 means for optimizing the volume irradiated by the laser beam, PA1 a unique cell within a capillary, the cell receiving a solute containing at least one unknown fluorescent substance at the laser wavelength, PA1 means for collecting the fluorescence emitted by the fluorescent substance, PA1 means for filtering the collected fluorescence, PA1 photomultiplier tube means for processing the filtered and collected fluorescence, and means for analyzing signals generated by the photomultiplier tube means and for displaying results of the analysis, wherein the cell employs controlled geometric contours to prevent laminar mixing and turbulence during liquid flow, and collecting means include high numerical aperture means. PA1 emitting a laser beam towards a capillary including a cell through which a solute flows containing at least one unknown fluorescent substance at the laser wavelength, PA1 collecting the fluorescence emitted by the fluorescent substance, PA1 filtering the collected fluorescence, PA1 transmitting the filtered and collected fluorescence to photomultiplier tube means, and PA1 analyzing signals generated by the photomultiplier tube means and displaying results of the analysis, wherein the emitting step and the collecting step are achieved collinearly. The emitting step includes a step for concentrating the laser beam in a ball lens and directing the resulting diverging beam into the ovoid cell. The collecting step includes the same ball lens, which has a very high numerical aperture. The ovoid cell has controlled geometric contours to prevent laminar mixing and turbulence during solute flow.
Previous techniques, which include Laser-Induced Fluorescence (LIF) detection with HPLC and CE techniques, together with narrow bore silica capillary have been successfully employed to assay samples whose molecular structure is relatively small.
HPLC and CE techniques, associated with UV-absorption detectors are widely used for the identification of a wide range of compounds in unknown substances. Nevertheless, UV-absorption detectors are limited in terms of sensitivity. To identify very low concentrations of substances, the LIF detection is therefore necessary.
Laser fluorimetry is very different from arc lamp fluorimetry and has some main characteristics, which are advantageous with a small volume detector. The produced fluorescent radiation is directly proportional to the intense laser excitation light. The amplitude stability of the laser also helps to minimize background noise level.
With LIF detections at least four problems have to be solved:
The fluorescence dF emanating from a volume dV is given by: EQU dF=c .epsilon. .phi.(P/S) dV
where
To get the highest fluorescence level, the excited volume containing the fluorescent molecules must be as large as possible.
Gordon U.S. Pat. No. 5,061,361! discloses a method and apparatus for increasing UV-absorption detector sensitivity in a Capillary Zone Electrophoresis detector. The detection means comprise a unique cell on the capillary presenting a dilated zone in the detection area, that employs controlled geometric contours to prevent laminar mixing and turbulence when using electro-osmotic pumping or pressure pumping. This so-called "enlarged capillary cell (or ovoid cell)" in which an unknown substance is migrating is subjected to an ultraviolet light. Part of the light is absorbed by the unknown substance.
Schrader U.S. Pat. No. 4,714,345! specifies (column 1 line 15-20) that "The theorical discussions (for Raman spectrometry) show that the LASER radiation has to be focused and the liquid sample has to be arranged at the focus."
Hlousek U.S. Pat. No. 5,037,199! discloses a cell assembly for use in spectrophotometric analysis or detection on column of a substance within a small sample volume. A first ball lens can be used for transmitting a non coherent light beam which excites a sample, while another ball lens is used for collecting the fluorescence. The exciting beam is arranged to be focused inside the capillary, and an index matching fluid may be used to facilitate the light transmission in the capillary bore.
Buttner and Beck EP 0 634 651 A1! propose a variant of an enlarged capillary for a CE system with UV-absorption detector or conventional fluorimeter, in which the fluorescence induced by a non coherent light is collected orthogonally, and the excitation beam is focused in the enlarged end part of the capillary.
The mode of focusing used in the example cited above is not compatible with a large illuminated volume required for laser-induced fluorescence.
Hernandez 1! 2! teaches that a high numerical aperture is required to collect the highest level of fluorescence on a capillary and to have the lowest background noise, and shows the evolution of the fluorescence versus the internal diameter of the capillary for a high numerical aperture objective. The intensity of fluorescence features a high increase for very low diameters (&lt;15 .mu.m), then stays nearly constant, and then decreases for higher diameters.
The use of a cell with controlled geometric contours prevents laminar mixing and turbulence which are necessary to maintain the best separation efficiency.
The main causes of light scattering in laser-induced fluorescence are:
In a collinear arrangement, these disadvantages may be overcome, since Raman back-scattering is avoided in the excitation axis 1! (compared to its nearly maximum level in a direction orthogonal to the incident light). The other causes may be mastered by adjusting properly the light paths and placing at appropriate location in the fluorescence light path spatial filter or diaphragm of selected diameter (0.5 to 1.5 mm).
The objective of this invention is to propose a laser-induced fluorescence detector device, for HPLC or Capillary Electrophoresis, with an increased sensitivity while employing capillaries having an enlarged capillary detection cell with an internal diameter substantially greater than 15 .mu.m and a sapphire ball lens, allowing the highest irradiated volume and the best fluorescence collection efficiency.